Seagrass nutrient uptake influenced by below-ground sulphide: a split-chamber hydroponic experiment.

Brief description

Rhizome sections of Halophila ovalis were collected from the Swan River Estuary at Pelican Point (31°59 S, 115°49 E) in January 2006 and were planted into a hydroponic, split-chamber experiment with constant water flow, designed to only expose below-ground seagrass biomass to sulphide. Sulphide concentrations were maintained at 0, 0.25 and 1 and 4 mM S2- and uptake of seagrass measured by removal of phosphate and ammonium from below-ground water. It was hypothesised that exposure of Halophila ovalis to below-ground sulphide would inhibit the capacity for ammonium and phosphate uptake and photosynthetic efficiency would be reduced.

Lineage

Maintenance and Update Frequency: notPlanned

Statement: Rhizome sections (with 2 fully expanded leaf pairs) of Halophila ovalis were collected from the Swan River Estuary at Pelican Point (31°59 S, 115°49 E) in January 2006 and were planted out into trays of acid washed quartz sand submerged in aerated aquariums filled with seawater. Plants were cultured in controlled temperature rooms at 20 °C (12 hr light : 12 hr dark) and received ~300 umoles of photons m-2s-1 PAR, which is above the saturating irradiance for H. ovalis (Hillman et al. 1995). Seagrass rhizome sections were acclimatised in culture for 2 weeks prior to the experiment. Rhizome sections were placed in to split-chambers (see thumbnail), and cultured in a continuous flow hydroponic setup. Watertight seals around petioles, made with a combination of Terastat VII (a waterproof plasticine type material) and melted paraffin wax, effectively separated roots and rhizomes from the above-ground biomass. Effectiveness of the water-tight seal was tested by introduction of a dye into belowground chamber. A low oxygen environment for water in the below-ground chamber was created by bubbling nitrogen gas in seawater for ~1 hour prior to Na2S addition. Below-ground water was enriched with ~240 uM NH4+ and ~15 M PO43-, added as ammonium chloride and di-sodium hydrogen ortho-phosphate. A constant flow of seawater (5 mL min-1) to the below-ground chamber provided by a peristaltic pump (Pharmacia-P3) exposed roots and rhizomes to the sulphide treatment of 0.25 mM, 1.0 mM or 4.0 mM S2-. The 3 channel pump circulated water through below-ground compartments of three chambers at the simultaneously. This water was circulated out of the reservoir and then returned to the reservoir in a closed circuit. The total volume water in the closed system was 1 L, made up of 900 mL in the reservoir and 100 mL in the below-ground chamber. Replicates ran from 11.30 am to 8.00 am the following day, a total of 20.5 hours. There were four treatments but as only three could be performed on any one day, treatments were staggered and cycled so that by day 4, three replicates of each treatment had been performed. Six replicates per treatment were planned, but a pump fault on day 7 curtailed the experiment. Six replicates for the control (0 mM sulphide) and five replicates for all other treatments were completed.

Photosynthetic efficiency of photosystem II (Fv/Fm) of Halophila ovalis was measured with a diving-PAM (Waltz, Germany) at t = 0 hrs (11.30 am), t = 6.5 hrs (5.45 pm), and t = 20.5 hrs (8 am the following day) while plants remained fully submerged. Dark leaf clips were attached to the most mature leaves(2nd fully expanded leaf pair) and left in place during the experiment to minimize damage to fragile tissues. Leaves were dark acclimatised for 10 minutes prior to measurements of Fv/Fm. Below-ground water was removed from the reservoir of each replicate at the following seven time intervals: 0, 0.75, 1.75, 3, 4.5, 6.25 and 20.5 hours. These samples were then used to both monitor sulphide concentrations in seawater, and determine phosphate and ammonium removal from below-ground water. 15 mL was removed at each sampling interval, with 5 mL used for phosphate determination. The remaining 10mL of below-ground water sampled was made basic (pH >10) with a few drops of 10 M NaOH, and sulphide concentrations measured with an ion selective electrode (TPS sulphide). Sulphide of water circulating in the below-ground chambers was supplemented if necessary by further addition of stock sodium sulphide solution. Changes in volume were recorded to enable correct conversion of concentrations to amounts (umoles) of phosphate or ammonium removed from below-ground water.
Plant material was removed from the split-chambers after 20.5 hours exposure to sulphide and dissected into above- and below-ground plant parts. These were then dried to constant weight at 60 °C. Grains of sand attached to root hairs unduly biased the below-ground biomass, so above-ground biomass was used to proportionally estimate below-ground biomass used for measures of nutrient uptake. Sampled below-ground water was stored at -4 °C until analysis. Ammonia was determined from on a Skalar Autoanalyzer (modified Berthelot reaction) and phosphate by the ascorbic acid method (Murphy & Riley 1962). Sulphide interfered with the ascorbic acid method, producing a reddish-brown solution at high sulphide concentrations. This interference was accounted for by calibration with standard phosphate solutions made up at each of the sulphide concentrations of the treatments (i.e. 0.25 mM, 1 mM and 4 mM) and phosphate calibration curves determined at 882 nm on a Shimadzu UV mini 1240 UV-Vis Spectrophotometer. This method of managing interference of sulphide was reasonable, however error may be introduced by variation in sample sulphide concentration (as oxidized in air). For future experiments, it is recommended that sulphide is removed (e.g. by precipitation) before nutrient analysis.

Notes

Credit
Strategic Research Fund for the Marine Environment (SRFME)

Purpose
To highlight the inter-relationship of sediment biogeochemistry and nutrient dynamics of seagrasses.